As noted above, my PhD work aimed to build a layer-by-layer (LbL) assembly at the surface of calixarene-based SLNs. To protect the DNA in the cell against attacks and to help the SLN-DNA complex to cross the cell membrane, chitosan, a polycationic biocompatible polymer, also known for its ability to enhance cellular uptake, was used. Nault et al., reported on the LbL assembly of 4NH24C12-based SLNs with the successive addition of
plasmid DNA and chitosan. They monitored the additions of DNA and chitosan by -potential measurement. An increase was observed in the
-potential values upon addition of DNA and a decrease in the -potential confirmed the addition of chitosan and the formation of a LbL coating at the surface of the 4NH24C12-SLNs. The SLNs were titrated with increasing
amounts of DNA as shown in Figure 50. The -potential decreased, confirming the addition of DNA around the calixarene-based nanoparticle. The opposite behavior was observed in the presence of increasing amounts of chitosan. Independently of the DNA, the GC12-based nanoparticles were fully coated with DNA 4 mg L-1 of DNA and 2 mg L-1 of chitosan.
Figure 50. -potential titration of AT (), GC (), ATGC () in the presence of increasing amounts of GC12-based SLNs, in buffer solution (20 mM HEPES, 10 mM NaCl, pH 7.1) (left), and GC12-DNA complex with AT (), GC (),
ATGC () in the presence of increasing amounts of chitosan in buffer
solution (20 mM HEPES, 100 mM NaCl, pH 7.1) (right).
The formation of a LbL assembly at the surface of GC12-SLNs was followed by -potential measurements. The positively charged GC12-SLNs in the presence of increasing amounts of DNA become negatively charged. Upon addition of chitosan to a solution of GC12-SLNs loaded with DNA, the objects in suspension become positively charged. The -potential increases after the addition of chitosan confirms the adsorption of the polymer at the surface of the SLNs. GC12-SLNs were successfully loaded using the LbL assembly technique (Figure 51).
-60 -40 -20 0 20 40 60 0 2 4 6 -p o te n ti al [mV ] DNA [g mL-1] -40 -30 -20 -10 0 10 20 30 40 0 0.5 1 1.5 2 -p o te n ti al [ mV ] Chitosan [g mL-1]
Figure 51. -potential of GC12-SLNs in the presence of AT (), GC (), ATGC () in buffer solution LbL coating of GC12-SLNs with DNA and chitosan layers.
The suspension were destabilized after addition of seven layers around the SLNs for AT and ATGC and five layers for GC. These differences are likely due to the specific DNA sequence used in the experiments. Further polyelectrolyte layer additions have been shown to cause a decrease in the stability of the suspensions, certainly due to particle aggregation. DLS measurements were carried out after the addition of DNA and chitosan layers but high polydispersity index values were obtained, most likely due to the formation of larger aggregates. The addition of
plasmid DNA into a suspension of nanoparticles was monitored by
-potential. The titration curves of the amino calixarene derivatives and GC12 are shown in Figures S11-S16 (Chapter 7). The addition of the first layer of DNA to a 1NH24C12–SLNs lead to a destabilization of the system.
-50 -30 -10 10 30 50 0 2 4 6 8 -p o te n ti al [ mV ] number of layers
To fully coat the calixarene-based nanoparticles, DNA at a concentration of 3 mg L-1, 4 mg L-1, 4 mg L-1, 5 mg L-1 and 4 mg L-1 was required for 1,2NH24C12, 1,3NH24C12, 3NH24C12, 4NH24C12 and GC12 nanoparticles
respectively. The subsequent titration of the nanoparticles’ DNA complex with chitosan reveals that the 1,2NH24C12, 1,3NH24C12, 3NH24C12, 4NH24C12
and GC12 nanoparticles need to be incubated with 3 mg L-1, 3 mg L-1, 5 mg L-1, 5 mg L-1 and 6 mg L-1 of chitosan respectively.
4.4 Calixarene-DNA
Complexes:
Self-Assembled
Lipoplexes
Due to electrostatic interactions, cationic lipids spontaneously interact with DNA molecules in aqueous solution, resulting in the formation of so-called lipoplexes. Lipoplexes are bilayer structures prepared by mixing preformed cationic liposomes with DNA in aqueous solution or by mixing cationic amphiphile with DNA without preformation of liposomes. Electrostatic interactions between the phosphate backbone of the DNA and the cationic recognition moieties of the amphiphile are the driving forces of the lipoplex formation. To study the self-assembly for calixarene–DNA complexes as lipoplexes, cationic calixarene liposomes were prepared. Calixarene powders (GC12 or the amino-calix[4]arene derivatives) were mixed at 40°C for 120 minutes in nanopure water or in buffer solutions. Four buffer solutions were tested: sodium phosphate buffer pH 7.5, HEPES buffer pH 7.05, Cacodylate buffer pH 7.1 and Tris-HCl
buffer pH 8.8. After 120 minutes the insoluble calixarene GC12 formed a clear solution.
Surprisingly, all the amino derivatives remained insoluble in aqueous solution in those conditions. The nano-suspension formed was characterized by -potential. The results are depicted in Table 8.
Table 8. -potential values of GC12-DNA lipoplexes in different conditions.
Buffer
-potential
GC12 liposomes GC12 liposomes mixed
with DNA at 20°C GC12-DNA lipoplex formed in-situ at 40°C Nanopure water + 45 ± 5 mV + 52 ± 6 mV + 58 ± 23 mV Phosphate -14 ± 11 mV -15 ± 9 mV - HEPES + 50 ± 12 mV - - Cacodylate + 67 ± 21 mV + 105 ± 17 mV +105 ± 9 mV Tris-HCl - - + 71 ± 8 mV
In water, HEPES buffer, and cacodylate buffer, -potential values of GC12 self-assemblies were measured to be + 45 ± 5 mV, + 50 ± 12 mV, + 67 ± 21 mV respectively. Positive -potentials suggest that the guanidino functions are pointing outward the nanoparticle. In phosphate buffer solution, a negative -potential was measured. This was attributed to the interaction of the phosphate ions with the guanidine functions at the surface of nanoparticles. In the Tris-HCl buffer, -potential distribution contained multiple peaks. It is likely that the positively charged GC12
thereby increasing the distance between the calixarene molecules and different species are measured in solution. Those preformed liposomes
were mixed with DNA for 30 minutes, 1.6 g, at room temperature. To investigate the in-situ formation of lipoplexes, GC12 powder was mixed
with DNA in nanopure water or in buffer at 40°C for 30 minutes. Both systems – the preformed GC12 nanostructure mixed with DNA at 20°C and the in-situ formation of GC12-DNA lipoplexes at 40°C – were analyzed by DLS and -potential. High polydispersity indexes were measured with large nanoparticle diameters. This was attributed to the presence of salt that may influence the stern layer around the nanoparticle. Discussion on these values will be highly speculative. In water and cacodylate buffer, as shown in Table 8, positive -potentials were measured for GC12-DNA complexes for both systems. GC12-DNA complexes prepared using preformed liposomes in water and cacodylate exhibited positive -potential values of + 52 ± 6 mV and + 105 ± 17 mV respectively. The GC12-DNA complexes
formed in-situ at 40°C exhibited similar positive -potential values of + 57 ± 23 mV and + 105 ± 9 mV in water and cacodylate buffer respectively.
Those -potential values were higher than the-potential values of + 45 ± 5 mV, + 67 ± 21 mV obtained for GC12 liposomes without DNA in
nanopure water and cacodylate buffer respectively. Multiple peaks could be measured for lipoplexes prepared with the preformed liposomes at 20°C in Tris-HCl buffer. This was attributed to a different organization of the self-assembly. A positive -potential of + 71 ± 8 mV was measured for GC12-DNA lipoplexes formed in-situ in Tris-HCl buffer. In HEPES and phosphate buffer, the addition of DNA disorganized the system and
negative or no -potential could be measured. It is known that cationic liposomes are destabilized upon addition of increasing amounts of DNA. Indeed, the liposomes might even release the DNA in solution at a certain lipid/DNA ratio.8-9
Conclusions
In this chapter, the ability of GC12 and of the amino calix[4]arene derivatives to self-assemble in aqueous solution as nanoparticles was demonstrated. DLS and AFM confirmed the presence of round nanoparticles in solution. Positive -potentials were measured, confirming the presence of the recognition functions at the surface of the nanoparticles. Interaction with DNA was studied by fluorescence displacement assay and revealed that 1NH24C12, 1,2NH24C12, 1,3NH24C12
and 3NH24C12 cannot displace the intercalating dye from the double-
stranded DNA, most likely because the interactions are mainly driven by electrostatic interactions. On the other hand, 4NH24C12 and GC12
displaced up to 80 % of the ethidium bromide from the double helix. Circular dichroism spectra and isothermal titration calorimetry data confirmed that the interaction of both SLNs with DNA is not only driven by electrostatic interactions but also by a groove-binding mechanism. Indeed, the SLNs interact with AT-DNA via a minor groove-binding and with GC via a major groove mechanism.
The calixarene-based nanoparticles 1,2NH24C12, 1,3NH24C12,
3NH24C12, 4NH24C12 and GC12 were coated with several layers of
DNA-chitosan using the layer-by-layer technique. In addition, the ability of GC12 to form lipoplexes in aqueous solutions was investigated. GC12 can form lipoplexes in the presence of DNA in pure water or in buffer solution.
References
1. Shahgaldian, P.; Da Silva, E.; Coleman, A. W.; Rather, B.; Zaworotko, M. J., Para-acyl-calix-arene based solid lipid nanoparticles (SLNs): a detailed study of preparation and stability parameters. International journal of pharmaceutics 2003, 253 (1), 23-38.
2. Sansone, F.; Dudič, M.; Donofrio, G.; Rivetti, C.; Baldini, L.; Casnati, A.; Cellai, S.; Ungaro, R., DNA Condensation and Cell Transfection Properties of Guanidinium Calixarenes: Dependence on Macrocycle Lipophilicity, Size, and Conformation. J. Am. Chem. Soc. 2006, 128 (45), 14528-14536.
3. Montasser, I.; Fessi H.; Coleman, A. W., Atomic force microscopy imaging of novel type of polymeric colloidal nanostructures. Eur. J. Pharm. Biopharm. 2002, 54, 281-284.
4. Kypr, J.; Kejnovská, I.; Renčiuk, D.; Vorlíčková, M., Circular dichroism and conformational polymorphism of DNA. Nucleic acids research 2009, 37 (6), 1713-1725.
5. Herrera, J. E.; Chaires, J. B., A premelting conformational transition in poly (dA)-poly (dT) coupled to daunomycin binding. Biochemistry 1989, 28 (5), 1993-2000.
6. Privalov, P. L.; Dragan, A. I.; Crane-Robinson, C.; Breslauer, K. J.; Remeta, D. P.; Minetti, C. A., What drives proteins into the major or minor grooves of DNA? Journal of molecular biology 2007, 365 (1), 1-9.
7. Yuan, Z.; Andrew, S.; Leaf, H., In Vivo Gene Delivery by Nonviral Vectors: Overcoming Hurdles? Molecular Therapy 2012, 20 (7), 1298-1304.
8. El Ouahabi, A.; Thiry, M.; Pector, V.; Fuks, R.; Ruysschaert, J. M.; Vandenbranden, M., The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids. FEBS letters 1997, 414 (2), 187-192.
9. Elouahabi, A.; Ruysschaert, J.-M., Formation and intracellular trafficking of lipoplexes and polyplexes. Molecular therapy 2005, 11 (3), 336-347.
5
Transfection Tests
The transfection experiments performed with calixarene-based SLNs coated with alternate layers of DNA and chitosan and with GC12-DNA lipoplexes are discussed in this chapter.To assess the possibility of transfecting mammalian cells, calixarene- based SLNs (1,2NH24C12, 1,3NH24C12, 3NH24C12, 4NH24C12 and GC12)
covered with alternate layers of DNA and chitosan have been investigated for their ability to deliver DNA into Chinese hamster ovary cells (CHO). The transfection was followed by fluorescence of a red fluorescent protein (DsRed2) for which the loaded DNA codes. The commercially available transfection agent Lipofectamine was used as a positive control.
Naked plasmid DNA was used as negative controls in the different aqueous solutions. Surprisingly, after 48 hours of transfection, no fluorescence could be detected using the calixarene-based SLNs coated with the LbL technique as a DNA carrying system. As Nault et al., were able to transfect MDCK (Madin-Darby canine kidney) cells using the 4NH24C12-
SLNs coated with several layers of DNA-chitosan, these results could be attributed to the use of different cells and different DNA for the transfection experiments.
The ability of the GC12-DNA lipoplexes to transfect DNA into the CHO cells was also investigated. The fluorescence microscopy images obtained after 48 hours incubation are presented below in Figure 52.
Figure 52. Fluorescence microscope images of CHO cells 48 hours after the
incubation with (A) Lipofectamine, (B1-F1) GC12-DNA lipoplexes prepared with preformed GC12 liposomes: (B1) in nanopure water, (C1) in sodium phosphate buffer (D1) in Hepes buffer, (E1) in cacodylate buffer, (F1) in Tris-HCl buffer; (B2-F2)
GC12-DNA lipoplexes prepared in-situ: (B2) in nanopure water, (C2) in sodium phosphate buffer (D2) in Hepes buffer, (E2) in cacodylate buffer, (F2) in Tris-HCl buffer. Imaged with 10x objective.
Figure 52 shows that GC12-DNA complexes in phosphate buffer solution (C1 and C2) were not capable of transfection. These results are
consistent as GC12-DNA lipoplexes are negatively charged in phosphate buffer, therefore the GC12-DNA complex could not cross the negatively charged cell membrane. For GC12-DNA lipoplexes formed in nanopure water (preformed liposomes or formed in-situ), only a few healthy cells could be observed (Figure 52, B1 and B2). In HEPES or cacodylate buffer,
a few healthy fluorescent cells and fluorescent aggregates could be observed (Figure 52, D1 and E1). The fluorescence increased using
lipoplexes formed in-situ in HEPES or cacodylate buffer, but the presence of aggregates was more pronounced (Figure 52, D2 and E2). It is likely that
the system is toxic or the cell self-destructed due to the presence of an
unknown system. The lipoplexes in Tris-HCl buffer solution (Figure 52, F1 and F2) showed the highest fluorescence intensity, but also the largest
amount of aggregates. The aggregates observed might be attributed to the necrosis of the cell or the aggregate observed might be small fractions of the cell that agglomerate. In the conditions tested, GC12-DNA lipoplexes seem to be toxic for the cells, but GC12-DNA complexes are able carry and protect the DNA to the nucleus and the protein fluorescence is expressed:
the lipoplexes could reach the nucleus of the cell and deliver the DNA. The ability of lipoplexes to transfect cells appears to be related to the
Several parameters such as the temperature, the buffer solution or method of preparation in one step (in-situ formation of the lipoplexes) or two steps (first the liposomes and then the lipoplexes) seem to be crucial parameters that affect the transfection.
In this chapter, the ability of GC12-SLNs coated with several DNA-chitosan layers and GC12-DNA lipoplexes to deliver DNA into
mammalian cells was investigated. Surprisingly, calixarenes-based SLNs coated with several DNA-chitosan layers were unable to deliver the genetic information into the cells. All the lipoplexes tested, with the exception of GC12-DNA lipoplexes prepared in phosphate buffer, were able to deliver the genetic information into the cells and express the protein. In particular the lipoplexes prepared in Tris-HCl buffer showed the highest fluorescence but also the highest amount of dead cells. On the other hand, GC12 lipoplexes in nanopure water were able to transfect cells with less efficiency, but the system was also cell toxic. The results are promising for the development of GC12 lipoplexes with high transfection and low toxicity. Several parameters can be tuned, such as the size, the lipid/DNA charge ratio, and the use of lipids (e.g. DOPE, DOTMA) to improve transfection efficiency.
6
Conclusions and Outlook
This thesis investigated the synthesis of new cationic amphiphilic calixarenes, the study of their self-assembly in water as nanoparticles, and the assessment of the calixarene-based nanoparticles to be used as a carrier system for DNA.The first achievement was the synthesis of amphiphilic calixarenes in the cone conformation, bearing guanidinium or amine recognition moieties at the upper rim and four dodecyloxy chains as hydrophobic functions at the lower rim.
The investigation of the self-assembly of cationic calix[4]arene at the air-water interface showed that all the cationic calixarenes tested were able to form stable Langmuir monolayers on a pure water surface. The interaction of the amino-dodecyloxy-calix[4]arene derivatives (1NH24C12, 1,2NH24C12,1,3NH24C12, 3NH24C12 and 4NH24C12 and GC12) with
ATGC in the subphase revealed different behavior depending on the number of amine functions available to interact with DNA double helix. Indeed, the compression isotherms of 1NH24C12, 1,2NH24C12,1,3NH24C12
and 3NH24C12 measured in the presence of ATGC in the subphase leads to
an expansion of the calixarene monolayers. No clear expansion, but a change in the shape of the isotherm could be observed with 4NH24C12.
To study the influence of the recognition function on the interaction of the calix[4]arene monolayer with DNA duplexes sequence, GC12 or 4NH24C12 monolayer isotherms were performed on three oligonucleotides
sequences: AT, GC and ATGC. The interaction of GC12 and 4NH24C12
monolayer at the air-water interface with the oligonucleotides (AT, GC and ATGC) in the subphase showed differences in interaction of the guanidinium and the amino functions with DNA molecules present in the subphase. Indeed, the compression isotherms of GC12 on a DNA subphase revealed a more important expansion of the monolayer than in the case of 4NH24C12 in the same conditions for all DNA tested. This was attributed to
the ability of guanidinium functions of GC12 to form two hydrogen bonds while the amine functions can form one hydrogen bond with the
Moreover, the interaction with the Langmuir monolayers is dependent on the DNA sequence. Different behaviors could be observed in the presence of AT, GC or ATGC.
The ability of GC12 and of the amino calix[4]arene derivatives to self-assemble as nanoparticles was investigated in nanopure water. GC12, 4NH24C12 and 3NH24C12 form stable solid lipid nanoparticles in nanopure
water. 1NH24C12, 1,2NH24C12 and 1,3NH24C12 in the non-protonated form
cannot form stable nanoparticles, but after protonation of the amine function, 1NH3+4C12, 1,2NH3+4C12 and 1,3NH3+4C12 formed stable
suspensions.
The interaction of the calixarene-based nanoparticles with the oligonucleotides ATGC was investigated by fluorescence displacement assay using ethidium bromide as intercalating agent. A slight decrease in the fluorescence intensity could be observed upon addition of 1NH3+4C12,
1,2NH3+4C12, 1,3NH3+4C12 and 3NH24C12 –based nanoparticles. These results
were attributed to the impossibility for the calixarene-based nanoparticles to unwind the double-strand of the DNA and to release the ethidium bromide in solution, most probably because the interaction with DNA double helix is mainly electrostatic. In the case of GC12-SLNs and 4NH24C12-SLNs, up to 80 % of the ethidium bromide was displaced for the
To investigate the influence of the DNA sequence in the binding capacity, the fluorescence displacement assay was performed in the presence of the 30-mer DNA AT and GC. The results suggested that GC12-SLNs and 4NH24C12-SLNs displaced the intercalating dye for all DNA
tested.
It was shown that the binding was not purely based on electrostatic interactions. In addition, the fluorescence displacement assay revealed a slight preference for AT-DNA for both systems.
Circular dichroism experiments for GC12 and 4NH24C12 showed that
the SLNs interact with DNA molecules and that those interactions caused a B-to-A-like transition for GC and ATGC and no transition in AT-DNA conformation. After the interaction, DNA duplexes remained in B’-form. The binding mechanism of GC12 and 4NH24C12-SLNs with DNA was
investigated with ITC. The ITC results suggested that the binding with AT proceeded via a minor groove mechanism while the binding to GC occurred mainly via its major groove for both systems (GC12-SLNs and 4NH24C12-SLNs).
The present work aimed at building an LbL assembly at the surface of calixarene-based nanoparticles to deliver genetic information into the cells. The calixarene-based nanoparticles 1NH3+4C12, 1,2NH3+4C12,
1,3NH3+4C12, 3NH24C12 and 4NH24C12 andGC12 were successfully modified
To go further in the study of the self-assembly properties of calix[4]arenes in the presence of DNA, lipoplexes were formed with GC12 calixarenes. No lipoplexes could be prepared with the amino calixarene derivatives. The SLNs and lipoplexes systems were used to deliver genetic information.
The results showed that the SLNs modified by the LbL technique could not transfect DNA in the condition of the experiments conducted. On the other hand, the lipoplexes formed in pure water or in buffer were able to deliver DNA into the cell but these systems were toxic for the cells in those conditions.
The self-assembly and DNA-binding studies of calixarene nanoparticles allow a better understanding of the mechanism of interaction between the recognition moieties at the surface of the nanoparticles and the DNA molecules. Considering the broader impact of research on nanoparticle as DNA carrier system, these results might be